Temperature and feeding frequency impact the survival, growth, and metamorphosis success of Solea solea larvae

Human-induced climate change impacts the oceans, increasing their temperature, changing their circulation and chemical properties, and affecting marine ecosystems. Like most marine species, sole has a biphasic life cycle, where one planktonic larval stage and juvenile/adult stages occur in a different ecological niche. The year-class strength, usually quantified by the end of the larvae stage, is crucial for explaining the species’ recruitment. We implemented an experimental system for rearing larvae under laboratory conditions and experimentally investigated the effects of temperature and feeding frequencies on survival, development (growth), and metamorphosis success of S. solea larvae. Specific questions addressed in this work include: what are the effects of feeding regimes on larvae development? How does temperature impact larvae development? Our results highlight that survival depends on the first feeding, that the onset of metamorphosis varies according to rearing temperature and that poorly fed larvae take significantly longer to start (if they do) metamorphosing. Moreover, larvae reared at the higher temperature (a +4°C scenario) showed a higher incidence in metamorphosis defects. We discuss the implications of our results in an ecological context, notably in terms of recruitment and settlement. Understanding the processes that regulate the abundance of wild populations is of primary importance, especially if these populations are living resources exploited by humans.


Abstract:
Human-induced climate change impacts the oceans, increasing their temperature, changing their circulation and chemical properties, and affecting marine ecosystems. At the current emission rate, the 1.5°C threshold will be exceeded by 2030 to 2052, and a 3-4°C temperature increase is predicted by 2100. Further, future projections performed within the Intergovernmental Panel on Climate Change (IPCC) context indicate a significant global reduction of primary production with critical consequences on fisheries and marine biodiversity. The flatfish species Solea solea , known as the common sole, is a crucial benthic and commercial species. Like most marine species, sole has a biphasic life cycle, where one planktonic larval stage and juvenile/adult stages occur in a different ecological niche. The year-class strength, usually quantified by the end of the larvae stage, is crucial for explaining the species' recruitment. We implemented an experimental system for rearing larvae under laboratory conditions and experimentally investigated the effects of temperature and feeding frequencies on survival, development (growth), and metamorphosis success of S. solea larvae. Specific questions addressed in this work include: what are the effects of feeding regimes on larvae development? How does temperature impact larvae development? Our results highlight that survival depends on the first feeding, that the onset of metamorphosis varies according to rearing temperature and that poorly fed larvae take significantly longer to start (if they do) metamorphosing. Moreover, larvae reared at the higher temperature (a +4ºC scenario) showed a higher incidence in metamorphosis defects. We discuss the implications of our results in an ecological context, notably in terms of recruitment and settlement. Understanding the processes that regulate the abundance of wild populations is of primary importance, especially if these populations are living resources exploited by humans.

Plos one
The following letter is to submit the manuscript entitled: "Temperature and feeding frequency impact the survival, growth, and metamorphosis success of Solea solea larvae by Adriana Sardi, Marie-Laure Bégout, Anne-Laure Lalles, Xavier Cousin, and Hélène Budzinski.
This work is part of a larger project that aims to understand the synergistic impact of increased temperature and contaminant exposure on physiological responses of the common sole Solea solea with an emphasis on larval stages. Our project evaluated the combined effects of PFOS contamination and temperature rise in S. solea larvae. For that, we simultaneously developed two approaches, experimental and modeling. The experimental approach included the development of a protocol allowing quantifying lifehistory traits during ontogenesis at the individual level. The modeling first focused on calibrating a Dynamic Energy Budget (DEB) model for S. solea and S. senegalensis. Further, the PFOS exposure data is for calibrating a DEBtox model, an ecotoxicological extension from DEB models that allows determining contaminants' physiological mode of action, i.e., how a pollutant interferes with energy budgets that fuel physiological reactions. Fellow researchers and I expect to use the models to predict the future impact of temperature, food availability, and chemical toxicity in fish early-life-history traits.
The results presented in this manuscript are only part of the project's results and are the control treatments regarding PFOS contamination. We propose working within microplates as a housing system to rear the larvae. This method presented several advantages, including monitoring the growth of larvae at the individual level and reducing mortality. Another clear advantage of working with microplates was for the contaminant exposure, as it guarantees even contaminant exposure and increases the number of replicates.
The work we submit for publication in Plos One fills a knowledge gap that aquaculture research has only partially grasped, specifically by exploring the interaction of temperature and food availability simultaneously. We put forward the protocol conducted as a simple test for investigating fish larvae's vulnerability to global changes.
Best wishes,

Adriana E. Sardi
Cover Letter 2 (if they do) metamorphosing. Moreover, larvae reared at the higher temperature (a +4ºC scenario) 30 showed a higher incidence in metamorphosis defects. We discuss the implications of our results in 31 an ecological context, notably in terms of recruitment and settlement. Understanding the processes 32 that regulate the abundance of wild populations is of primary importance, especially if these

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The Intergovernmental Panel on Climate Change (IPCC) has been, since its first report back in underline that the consequences of global warming of 1°C are genuine, particularly the increased 43 occurrence of extreme weather events, the rise in sea level, and the decrease in Arctic sea ice (1).

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Following the release of the most recent report, leading author, Dr. Joeri Rogelj, highlights that this 45 report is likely to be the last one while there is still time to stay below the 1.5ºC threshold (The 9/08/2021). At the current emission rate, the 1.5°C threshold will be exceeded by 2030 to 2052, 48 and under the IPCC RCP 8.5 scenario, a 3.7°C global mean surface temperature increase is 49 predicted by 2100 (2). In this context, future projections indicate a significant global reduction of 50 primary production with important consequences on fisheries and marine biodiversity (3,4).

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Most marine species, including teleost fishes, have a multiphasic life cycle, where one -or multiple-52 planktonic larval stages and juvenile/adult stages occur in a different ecological niche (5). The 53 complexity of life cycles with multiple and distinct phases is thought to promote higher dispersion of 54 individuals due to oceanic currents followed by reduced predation and justified by access to a 55 larger food sources (5). However, having different niche on different life stages also means that 56 different stages will be exposed to different scenarios, making potentially harder the transition from 57 one stage to another.

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Compared to any other life stage of a marine fish species, individuals at their larval stage will have 59 the highest potential for growth, weight-specific metabolic rates, natural mortality rates, and the highest sensitivity to environmental stressors (6,7). Early stages have strict environmental    the end of metamorphosis, juvenile soles are ready to change from pelagic to benthic lifestyle to colonize shallow coastal waters in estuaries and bays (15). After settlement, juveniles stay for kilometers depending on location) for reaching spawning grounds, and they eventually come back closer to the shore out of reproduction season to reach feeding grounds (11). To summarize, three to determine how habitat quality affects population dynamics.

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As an r strategist species and a batch spawner, S. solea is characterized by a high fecundity. The spawning of hundreds of thousands of eggs per kilo of female with overall low survival rates is 102 triggered by changes in water temperature trigger (13).

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The reproductive success of soles -quantified as the number of eggs, larvae, or juveniles that 104 survive and settle in coastal nursery areas-widely depends on hydrodynamic processes, and it is 105 regulated by environmental factors, among them, temperature (16). While ecological processes 106 acting in nursery areas can affect year-class survival, these are generally considered less 107 important than those influencing larval stages. This assumption relies on the fact that nurseries are stable habitats that provide suitable conditions for the survival of juveniles (16).
Understanding the processes that regulate fish recruitment are of primary importance to assess

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We now generally acknowledge that no single process, mechanism, or factor is responsible for 119 recruitment variability but that many processes may act together over the entire egg to the pre-120 recruit juvenile period (19). Among the multiple processes involved, dominant recruitment large size and fast development is better as it reduces predation pressure and improves survival (19). enough food to compensate for the rise in energy demands a higher metabolism implies. In the 128 wild, reduced food availability or starvation would slow metamorphosis and increase predation risk, 129 thus reducing larval survival.

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In this work we experimentally investigated the combined effects of temperature and feeding 131 frequencies on the survival, development (growth), and metamorphosis success of S. solea larvae.

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Our objective was to disentangle the effects of food availability and increased temperatures -

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In all our experiments, we used 24-well polystyrene microplates with a cover as a housing system.  To ensure the viability of using microplates as a housing system, we ran a pilot experiment in April 148 2019. The objectives were to test the rearing system -particularly the survival of larvae-and to in the Netherlands (Zeeschelp, Kamperland). Sole adults naturally spawned in several batches under the stereomicroscope (Olympus, SZX9) and transferred to glass bottles containing clean 154 and filtered (GF-C 0.45µm, Millipore) natural seawater at a salinity of 30.

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Hatching occurred during the weekend of 27 th and 28 th of April (between 4-and 5-days post-158 fertilization, depending on the batch).

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Two days post-hatching (dph), 72 larvae were transferred to 24-well polyethylene microplates 160 (Sigma-Aldrich, CLS3527-100EA). Before transferring, we prepared the plates by adding 1950 µl 161 of the water media. Larvae were gently pipetted on a volume of 50 µl and transferred to the wells.

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We used a 200 µL capacity micropipette with a tip cut to transfer the newly hatched larvae and 163 avoid damage to the entering larvae.

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All microplates were placed within an environmental chamber and incubated on the same 165 conditions described above. Survival, malformations, behavior, and metamorphic stages, were 166 monitored daily using an Olympus SZX9 stereomicroscope. The experiment lasted for 33 days.

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Water was changed every other day by carefully removing as much as possible of the water 168 (~1800 µl) while moving the tip away from the larvae to avoid suctioning it.  Please refer to Table S1 to know the exact amount of artemias given to the larvae daily until the end of the experiment.
In 2021, we obtained fertilized eggs from the same broodstock, and to determine the timing for starting the temperature treatments, we ran a second pilot experiment, which was immediately to the 28 th of April, and eggs arrived at the Ifremer Palavas-les-Flots, France the 29 th of April 2021.

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Eggs employed in the experiment spawned the 8 th of May, and arrived at Ifremer the 12 th of May.

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In both occasions, fertilized eggs were sorted and transferred to glass bottles containing freshly 183 prepared artificial seawater at a salinity of 30 and a temperature of 16ºC. Artificial seawater was 184 prepared by diluting 30 g of salt (Instant Ocean) in type II water, and we adjusted salinity to 30 185 using a salinometer. Further, we filtered the water using GF-C 0.45 μm to remove non-dissolved 186 particles.

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For the pilot experiment we equally distributed larvae from different batches on the experimental 189 microplates while in the experiment all larvae were from the same spawning event (8 th of May).
Procedures for transferring larvae to microplates and monitoring survival and development were  The experimental design for pilot 2 and the experiment included two experimental factors, 196 temperature with two levels, 16ºC and 20ºC, and a feeding (density in the pilot and frequency in 197 the experiment) factor, which was assumed to be a proxy for food availability (Fig. 1a). The last 198 factor included three levels: high food, medium food, and low food, from now on indicated in the 199 text as HighFood, MediumFood and LowFood, respectively. The main differences between the two 200 pilots and the experiment are summarized in Table 1.

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In each experiment, two groups of microplates were designed. The first group, called the 202 experimental group, consisted of 3 microplates and 72 larvae. Each temperature treatment 203 included three feeding regime levels. Larvae from the experimental group were planned for included 3 additional microplates and 72 larvae, with the same treatments as the experimental 206 group. These larvae were planned for monitoring changes in length and dry weight along the 207 experiment. For that, a total of four larvae per plate were sacrificed every week.

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Effects of temperature and food availability

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To study the effects of temperature on sole larvae growth and development, we incubated larvae 210 at 16ºC, an optimal temperature for S. solea larvae development, and at 20ºC, a + 4ºC condition

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In pilot 2 larvae were directly placed in the environmental chamber at 20ºC following hatching and 215 the food availability treatments consisted in different food densities. This pilot experiment allowed 216 us to improve the method of larvae acclimation to the 20ºC temperature treatment. In the 217 experiment larvae were acclimated with a gradual temperature increase (Fig. 1b). For this, we 218 increased the temperature of the environmental chamber by one degree every day from day 8 219 post-hatching, which corresponded to three days after the first meal or mouth opening day.

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The effects of food availability were approximated and tested by comparing larvae 222 metamorphosis, survival and growth from larvae reared with different feeding frequencies. To 223 feed recently hatched larvae we provided A0 Artemia salina (less than 24h old individuals).

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Because recently hatched larvae are too small to eat enriched A1 artemias, we decided to 225 provide unenriched food along the whole experiment.

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In the second pilot, the quantity of A. salina provided from mouth opening day differed between 227 treatments, with HighFood treatment receiving an amount of artemia assumed to be ad libitum.

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The amount of artemia ad libitum per age was obtained from our first pilot experiment. We 229 calculated the amount of artemias for the other two treatments representing 60% and 40% of the During the experiment, we fed equally all larvae every day during the first five days post mouth 233 opening. From 12 dph onwards, we changed the feeding frequency among treatments as it was 234 a simpler approach than altering the food density. HighFood larvae were fed 6 days a week, 235 while MediumFood and LowFood treatments were fed three times and twice a week, 236 respectively. On 12 dph, we provided around 15 artemias, and as in pilot 2 the amount of food 237 gradually increased along the experiment.

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These larvae were sacrificed and collected from the biometric group microplates described before.

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We euthanized the larvae and placed them one by one on a microscope calibration slide (grid 1 254 mm). Because larvae survival was lower than anticipated, from week 4 until week 7 of the 255 experiment, only length measurements without killing the larvae were taken. We did so by simply

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The testing design consisted of 2 factors orthogonal to each other, feeding density in experiment 1 274 or feeding frequency in experiment 2 (fixed, three levels, HighFood, MediumFood, and LowFood), 275 temperature (fixed, two levels, 16ºC, and 20ºC), and their interaction. If the interaction was not 276 significant, differences among feeding frequencies were retested for each temperature separately.

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For the PERMANOVA tests we used the package vegan (29) and significant terms in the model (α

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We determined the LT50 for each treatment, defined as the time for 50% of the individuals to die.

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For that, survival data of each treatment combination was fitted to a four parameters logistic curve, 290 using Prism (Graphpad).

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During the first days of the experiment, the number of surviving larvae decreased considerably, 295 being the larvae reared at 20ºC LowFood, the ones that died faster. Both temperature treatments 296 had the same decreasing tendency, and the lethal time for 50% of the individuals (LT50) happened 297 in average 2 days earlier in larvae reared at 20ºC vs. larvae reared at (Table 2). At 20ºC, the 298 lowest LT50 was obtained for the MediumFood treatment, which was significantly different from Low 299 and HighFood. No differences were detected between the LT50 obtained for larvae reared at 16ºC.

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As expected, the food treatments did not affect the first week's survival, when all larvae were fed 301 similarly and larvae still rely on their yolk reserves. Thus, the overall survival among treatments 302 was very similar during the first two weeks (Fig. 2). Larvae reared at 20°C had a period of 303 acclimatization that started at day 6 of the experiment (larvae aged 8 dph) when the temperature 304 was progressively increased by 1 degree per day, ending on day 10 (12 dph). The similar trends in 305 survival among treatments obtained during the first week of the experiment are most likely a result 306 of the acclimation period. However, from the second week onwards (from day 8 to 15 in Fig. 2),

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there is an important decrease in survival, with both temperature treatments following the same 308 trend, i.e., a sharp decrease in the survival rate for all treatments that stabilizes around day 18 (at 309 20 dph, Fig. 2). The lowest percentage for total survival was obtained for LowFood treatment at 310 20ºC (Table 2). For comparing the survival rates and their relationship to food availability, we fitted 311 linear regressions for the survival curves during the period where the decrease in survival was the 312 highest (Fig. 2, bottom). The results show that survival was significantly different between temperature treatments in the LowFood treatment (no overlapping on the 95% confidence 314 intervals).

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The interaction between temperature and feeding frequencies does not explain the observed 320 variation in the total length of larvae (Fig. 3, Table S3). However, the feeding frequency factor did 321 affect larval length independently (p-value feeding < 0.001) while temperature did not have a 322 significant effect on growth for, at least, the first 35 days following hatching.

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For the effect of feeding frequencies, a pairwise test showed that larvae from the HighFood 324 treatment were significantly longer than larvae fed two times per week (LowFood p-value < 0.01).

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No significant differences were observed between HighFood and MediumFood or MediumFood 326 and LowFood treatments.

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By the end of the experiment, the longest larvae were from the HighFood treatment reared at 20ºC, 328 accounting for an average length of 9.8 ± 0.6 mm ( Table 3).

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Given the high mortality we observed during the first 3 weeks (Fig. 2), we decided to stop sampling 330 larvae for quantifying the dry weight of larvae. Thus, there are no dry weight records during the 331 fourth, and fifth weeks of the experiment.

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Regarding dry weight data, none of the tested factors or their interaction explained the observed 333 variation (Fig. 4, Table S3).

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In the HighFood treatment alone, we observed the same pattern regardless of the rearing 335 temperature, showing a decrease in dry weight from day 8 to 15, followed by a steep increase in 336 weight towards day 22 and 35. For the other two feeding frequencies, data showed little variation from the start until the end of the experiment (Fig. 4). process at 19 dph. At 16ºC, the first larva that started metamorphosis was at 22 dph.

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A total of 5% of the surviving larvae showed abnormal metamorphosis, often involving the non-348 migration of the left eye (Fig. S2). All metamorphosis abnormalities were obtained in the HighFood 349 treatment at 20ºC, which was also the treatment with the highest proportion of individuals 350 metamorphosing.

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By the end of the experiment in HighFood, the average size of larvae at stage 4 was higher (but 352 not significant) for larvae reared at 20ºC (10.9 mm ± 0.4, n=8) vs. those reared at 16ºC (9.4 mm ± 353 0.3, n=3). Further, the first larva that finished metamorphosis were 10.1 mm long, and 9.4 mm for 354 20 ºC and 16ºC, respectively. Larvae in the MediumFood treatment did not complete 355 metamorphosis and the average size of stage 2 larvae when reared at 20ºC was slightly smaller 356 (7.11 mm ± 0.3, n=9) vs. those larvae reared at 16ºC (7.54 ± 0.2, n=8), which were still on stage 1.  and collaborators published a protocol where sole larvae were reared using 24-well microplates as within a system that allows rearing larvae individually is a great advantage. Using microplates as a

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The accentuated differences we obtained in the survival of control larvae between pilot and 384 experiment could be explained by the quality of the eggs. The same observation applies for 385 survival during the second pilot compared to the experiment (Fig. S4). Indeed, our observation 386 during the second pilot -later confirmed with data-was that very few larvae started feeding,

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indicating that the transition between endogenous to exogenous feeding was not successful for 388 most larvae, regardless of the treatments. In terms of reduced survival, following a 5 days delayed 389 first feeding, a work by Lagardère et Chaumillon (35) also obtained very low survival rate (1.3%) 390 after 22 days of experiment. At 33 dph, total survival in the pilot experiment was 81%, whereas, during the experiment, all treatments and temperatures considered were below 50% at the same 392 age.
Temperature changes affect physiology, gamete development, and maturation, typically ending in poor sperm and oocyte quality, albeit gamete quality is crucial for developmental success in the suitable egg-laying. First, the first spawning occurred late, followed by a cold wave that disrupted

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The critical decrease in survival occurring during the second week corresponds to the transition 404 from endogenous to exogenous feeding. The abundance of food plays an essential role in 405 determining the survival of first-time feeding larvae. This is because, after yolk exhaustion, fish 406 larvae must establish themselves as active feeders, or they risk starvation (37). Generally, the time 407 span from yolk exhaustion to starvation is temperature-dependent and species-specific. Effects of 408 feeding frequency in larvae survival have already been studied in several species including 409 Oreochromis niloticus, Lates calcarifer, and Heterobranchus longifilis (38). Atsé and collaborators 410 (2012) tested the effects of feeding rates (25%, 50%, 75%, and 100% of biomass) and feeding 411 frequencies (one meal per day either in the morning or in the afternoon, two meals per day, and 412 three meals per day) on survival and growth of Heterobranchus longifilis larvae over a rearing time 413 of 28 days. They showed that growth and survival were proportional to increasing feeding rate.

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Also, survival and growth were highest in larvae fed three times a day, and cannibalism was less 415 important with increasing feeding rates. All these results allowed them to conclude that optimal 416 conditions for rearing this species include a feed ration of 100% and a feeding frequency of three 417 meals per day.

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In the case of sole metamorphic larvae reared under laboratory conditions, density, ration, or 419 feeding frequency and the timing for the first feeding can influence the survival and growth of larval varies mainly according to species, size, and rearing conditions -notably temperature-and the ideal feeding rate and frequency generally decrease as the fish grows (39).
suggested for the Japanese flounder, the relatively higher mortality at 20°C could be explained 425 because the metabolic and energy costs are higher than that of sole reared at 16°C. As a result, 426 feeding becomes more difficult as the larvae have to spend energy, which they have little, to hunt, 427 particularly in those treatments where food availability is lower (37). 428 The protocol adjustments made within this work make it a much more accessible, reproducible and  The hypothesis that lower food availability will delay the start of metamorphosis while temperature 442 increase will fasted up growth was not refuted. However, our data also reflects the complexity of 443 interpreting these results as the effects of these factors are highly interconnected.

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In our experiment, the largest larvae and the faster growth rates were those from the HighFood 445 treatment reared at 20ºC, and the start of metamorphosis was affected by feeding frequency, with HighFood larvae starting it 14 and 16 days before MediumFood and LowFood treatments, rates, faster life histories -notably regarding metamorphosis, which is advanced in larvae reared at 473 20ºC-and higher natural mortality rates when food density is low.

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At higher temperatures, the metabolic rate of larvae is expected to increase, which means that fish 475 have higher basal energy demands and larval growth rates increase. Although responses vary considerably among species, mass-specific oxygen consumption in fish larvaea proxy of 477 metabolic rate-has been proven to increase with temperature (44). Regarding larval growth rates, 478 empirical observations show that growth rates tend to be approximately linear until the lethal upper 479 thermal limit is reached, at least in most species studied to date (45). The previous statement 480 contrasts with thermal reaction norms -continuous functions describing the relationship between an 481 environmental variable (e.g., temperature) and the phenotype expressed by a given genotype (46)-482 of growth in juveniles and adults, which usually decline well before the lethal thermal limit is 483 reached. The temperature range for the successful development of S. solea eggs is between 7 to 484 19°C, 10 to 23°C for larvae, and 7 to 27ºC for juvenile growth (41,47). Thus, under the worst RCP 485 scenario temperature predictions, we might expect that larval growth rates will tend to be 486 maintained, whereas effects on juvenile growth -such as shorter asymptotic lengths-might be 487 observed. Our results support the last, as the slopes for size and weight measurements in both the 488 HighFood and MediumFood treatments were similar for larvae reared at optimal and +4ºC (Fig. 4).

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Changes in the pelagic larval duration (PLD) and potential mismatch between hatching and the 490 zooplankton cycle timing are far more preoccupying effects of temperature increase than changes 491 in larvae and juvenile growth rates. Indeed, the times until yolk absorption, metamorphosis and 492 PLD of fish larvae are all negatively correlated with temperature (45), observations also evidenced 493 by our results. Because mortality is generally very high during the larval phase, faster growth and 494 shorter PLD at higher temperatures could positively affect larval survivorship (45). In our 495 experiments, the survival of larvae reared at 20ºC was higher than that of larvae at 16ºC for the 496 HighFood treatment, supporting the previous assumption.

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In this work we did not study the effect of temperature on spawning. Our results evidenced that 498 larvae reared at higher temperatures have a shorter PLD, suggesting that higher temperatures due 499 to climate change might favor larvae survivorship. However, during our pilot experiment we 500 observed massive mortality in all treatments, which could also be related to larvae missing their first feeding due to low prey densities or reduced capabilities in capturing prey (linked to egg result supports the critical period hypothesis in which first-feeding survival larvae define year-class 504 strength and fish recruitment success.

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Food availability is compulsory for sustaining higher development and growth in warmer waters. As 506 climate models predict an increase in water temperature and lower food availability, the potential 507 increase in survival probability of fish larvae due to faster growth and shorter PLD that reduces the 508 predation pressure (as proposed in the size/growth hypothesis) would be counterbalanced by the 509 increase in mortality during the critical period due to lower prey densities.

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Given that changes in phenology vary among functional groups -as evidenced by (49)  In this work, we experimentally tested the effects of food and temperature -two important environmental drivers-in sole metamorphosis success separately, but in natura, it is unlikely that could cause a mismatch between different trophic levels and negatively affect recruitment not only 533 by directly increasing mortality but also by extending the metamorphic period. Because the onset 534 of metamorphosis requires larvae to acquire a competent size, when conditions are not favorable 535 for growth, metamorphosis takes longer and renders larvae vulnerable to predation. On the 536 contrary, a higher temperature without food limitation could be an advantage as it would speed up 537 the metamorphosis process. However, there is still the risk of finding fully metamorphosed 538 juveniles outside nursery areas, and as such, still vulnerable to predation and in an environment 539 low in preys.

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Although the interaction between feeding and temperature was not statistically significant in both 541 experiments, it was the LowFood treatment at 20ºC, the treatment with the highest mortality.

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Larvae growing at higher temperatures have higher metabolic rates and higher energetic 543 demands, but they will die if food is unavailable to meet these demands.

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The objective of this study was to determine the impact of different feeding frequency regimes -as 547 a proxy for food availability-on common sole larvae development in the context of global warming.

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For this, we hypothesize that 1) feeding frequencies would impact the survival, growth, and 549 metamorphosis success of the larvae and 2) rearing temperature would increase the observed 550 effects by adding additional physiological stress to larvae reared at +4ºC.

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Accounting for our results, we cannot entirely accept the first hypothesis, as we did not find a 552 statistically significant relationship between survival and the tested treatments. However, we 553 observed significant effects of the feeding frequency factor on larvae growth (measured as length 554 and dry weight), and there were differences in the start and pace of metamorphosis. The more we 555 fed the larvae, the longer and heavier they were. Regarding the metamorphosis of the larvae, we and fed HighFood begun their metamorphosis ten days before Medium Food larvae and 17 days 558 before LowFood larvae. At 20°C, the start of metamorphosis was even faster, with HighFood 559 larvae beginning their metamorphosis 14 days before MediumFood ones, and 16 days before 560 LowFood larvae.

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Overall, the feeding frequency regimes impact growth and metamorphosis but not survival.

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However, this is only true if the larvae are fed ad libitum during the first week. During our second 563 pilot, the larvae were not fed until satiation, altering the transition from endogenous to exogenous 564 feeding and leading most larvae to die. Based on the experiment results, our hypothesis of feeding 565 frequency regimes altering growth and metamorphosis should be accepted.

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Summarizing, food availability impacts the growth and onset of metamorphosis in S. solea larvae, 567 while higher temperature advanced the onset of metamorphosis and increased the occurrence of 568 abnormalities.

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Future perspective 571 Experimentally testing the effects of any stressing factor is always a challenge, mainly because 572 rearing and keeping animals in laboratory conditions is already very stressful for most of them.

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Here we aimed at testing a user-friendly protocol for evaluating the effects of food availability and 574 temperature on the larval development of the common sole. We focused in larval phase, as there 575 is a substantial research library on sole juveniles, with less information linking larvae survival and 576 juvenile recruitment. Despite conditions being far from perfect, we propose a reproducible 577 experiment that provides insights into the health and survival of larvae exposed to conditions 578 mimicking future climate change scenarios. Recommended modifications for the proposed protocol 579 include 1) using 12-well microplates for providing a larger volume and area to larvae, and 2) 580 providing enriched food with higher nutritious value, which would allow identifying caveats of the 581 experiment related to energy while minimizing sources of bias such as fish behavior.

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Climate change impacts might not operate independently from toxicity effects from pollutants. We 583 consider that the protocol proposed here is suitable for replacing standard ecotoxicological testing last is the most frequently used bioassay for fish toxicity, supporting aquatic ecological risk 586 assessments and chemical management programs. However, this test guideline requires an 587 average post-hatch control survival of at least 75% in animals usually housed in the same 588 chamber. These conditions are fulfilled for some model fish species, which are often very robust 589 laboratory species that might have already undergone adaptations to laboratory conditions 590 (following multiple generation rearing), such as rainbow trout, zebrafish, Japanese medaka, and 591 fathead minnow. However, when the interest species is not listed in the recommended annex from 592 the OECD guidelines, the chances are that survival higher than 60% in control will not be met.

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There is a need for testing chemical toxicity in other less robust species, and one way to reach it is 598 by developing or adapting current methods for assuring higher survival rates. Individual housing in 599 microplate wells increased larvae survival, which is an appropriate experimental improvement.

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Standardizing a protocol that allows evaluating the effect of contaminants in this species is relevant 601 given its economic and ecological importance and its susceptibility to chemical and fishing 602 pressures.  Determining the timing for starting the temperature treatments. Setting the different food treatments.
Determining the combined effects of temperature and feeding frequencies in sole larvae Larvae exposed at 20ºC (age) Immediately after placing larvae in microplates (4-5 dph) Temperature increased gradually, 1 degree per day from day 8 posthatching (larvae at 20ºC from 11 dph onwards) Food availability at mouth opening day Ad libitum. The quantity of food provided and consumed per larvae each day was monitored (counted) Different food densities among treatments HighFood = 5 artemias MediumFood = 3 artemias LowFood = 2 artemias Same food density and feeding frequency during the first 7 days following mouth opening (10 artermias per larvae).
From 12 dph onwards, we changed the feeding frequency among treatments (with an increasing number of artemias 15 to 40) the following way: HighFood: fed every day MediumFood: fed every other day LowFood: fed twice a week 751